Over the past several years, new techniques in cryo-electron microscopy single particle analysis, a technique whereby images of identical protein complexes at many different angles are computationally combined to determine the 3D structure of the protein, have made it possible to achieve near-atomic resolution for protein complexes 500 kDa. In 2015, we published the structure of a complex between E. coli beta-galactosidase and the cell-permeant inhibitor phenylethyl beta-D-thiogalactopyranoside (PETG), at 2.2 Angstrom resolution, a study that showed not only specific interactions at the drug binding site, but also a large number of stable water molecules in the structure. In addition to beta-galactosidase, we have also applied these new techniques to a number of additional soluble protein complexes. Pushing the boundaries of both size and resolution, we published a collection of structures of metabolic enzymes: we determined the structure of the core region glutamate dehydrogenase (GDH) in its open state to 1.8 Angstrom resolution, the highest resolution cryo-EM structure yet reported; we determined the structure of lactate dehydrogenase (LDH), a 145kDa tetrameric complex, bound to a small molecule inhibitor, to 2.8 Angstrom; and we determined the structure of isocitrate dehydrogenase (IDH), at 97kDa the smallest complex determined to high resolution by cryo-EM, to 3.8 Angstrom resolution. The structures of these three complexes required a number of new methodological adjustments, including collecting data from areas of extremely thin ice, retaining only images with minimal sample movement, and using the latest high quality detectors. In collaboration with NCATS and several extramural academic institutions, we also undertook a structural study of p97, an AAA ATPase that has be challenging to study by crystallography due to its heterogeneous, highly flexible nature. We determined structures of p97 to high resolution in several distinct states, showing a that the complex cycles through a series of conformational changes during the ATPase cycle. In addition, our 2.3 Angstrom structure of p97 in complex with an allosteric small molecule inhibitor showed that the inhibitor functions by preventing the normal corkscrew-like motion of the D1 and D2 domains during ATPase activity, essentially inhibiting p97 activity as if the small molecule were a wrench in the works. Last year we emphasized the exciting possibility that cryo-EM could be used to derive de novo, high-resolution structural models of proteins in one or multiple functional conformational states, allowing the analysis of structures of a wide variety of biologically and medically relevant multi-protein complexes and membrane protein assemblies, which have historically represented the most challenging frontier in structural biology. This year, we have applied this technique to a variety of complexes, such as p97 as mentioned above, as well as the membrane proteins GluR, CorA, and others (mentioned in our membrane protein project). We expect that as we continue to drive this technology forward, cryo-EM analyses will increasingly provide novel, critical insight into the structures of a variety of historically intractable complexes, and information about the binding of small molecule and immunological therapeutics to these structures.